Disclosed is a novel device for detection of an analyte in a sample using a lateral flow assay. The device and method of the present invention may be easily and inexpensively assembled, and suitable for use by personnel with little specialized training. The device further provides for sanitary handling and disposal of biological samples. The device and method disclosed herein may be used with labels conventionally used with lateral flow assays such as colloidal metals, or may also be used with colorimetric or fluorescent labels that require instrumentation for detection.
Immunoassays
The present invention relates to assays utilizing test strips, in particular, a lateral flow immunoassay. The immunoassay, in general, is a sensitive technique used to measure levels of a substance using the reaction of an antibody or antibodies to its antigen. Immunoassays generally rely on binding of an antibody to an antigen. Monoclonal antibodies, in particular, are often used because such antibodies generally bind to only one site of a particular molecule. This specific binding enhances the specificity and accuracy of binding to a particular analyte. The antibodies used in immunoassays typically have a high affinity for the antigen such that a high proportion of the antigen binds to the antibody.
Immunoassays are powerful and versatile biomedical diagnostic tools that can be used, for example, to monitor drug and hormone levels in body fluids, diagnose infectious and autoimmune diseases, and diagnose and monitor treatment of cancer.
One analyte in particular that is ideally suited for detection using immunoassay techniques is influenza. Influenza is a highly contagious epidemic to pandemic acute viral respiratory disease caused by several genera of the Orthomyxoviridae family. Influenzavirus A and Influenzavirus B are the two genera most commonly associated with disease in humans. Influenza infection rates tend to be highest in pediatric populations, while serious complications from influenza disease are more common in the elderly. Clinical signs and symptoms begin after a 1-4 day incubation period and include cough, fever, myalgia and malaise. The clinical presentation of influenza can range from asymptomatic infection to fatal pneumonia. Influenza co-circulates with other respiratory pathogens; hence it is important to differentiate influenza from other respiratory diseases. Rapid influenza detection tests facilitate the more timely administration of antiviral drugs, which, in general, are of clinical benefit when administered within 48 hours of the appearance of symptoms. Not all antiviral drugs are effective against both influenza A and influenza B; therefore it is important to distinguish between the two.
Influenza A and B can be detected in human respiratory samples by a variety of methods including tissue culture, immunofluorescent assay and enzyme immunoassay. Tissue culture isolation remains the gold standard for the detection of influenza, yet the procedure can take up to 7 days to complete. Immunofluorescent antibody-based tests are moderately sensitive, yet highly dependent on specimen quality and preparation. The rapid detection of influenza using enzyme and microparticle-based immunoassays has become an important aspect of patient management in patients of all ages with acute respiratory disease due to influenza. Test results can be used to support data available from the patient's clinical evaluation and assist the physician in determining the course of action.
Immunoassay techniques typically employ a detectable label that permits the user to determine whether the analyte is present in the sample. The label can be conjugated to a particle such as an antibody that binds to the analyte (referred to herein as a first “binding reagent”). The type of label used may vary, and may include visually detectable labels as well as labels that require instrumentation for detection. Non-limiting examples of labels that can be used with immunoassay techniques include enzymes, radioisotopes, fluorescent tags, carbon particles, beads, or metal sol tags such as colloidal gold.
Lateral Flow Immunoassays
Lateral flow assays (or “flow-through” assays) are well known in the art and are described in Ching et al., U.S. Pat. No. 6,534,320, May et al., U.S. Pat. No. 6,228,660, Charlton et al, U.S. Pat. No. 5,989,921, Charlton U.S. Pat. No. 6,485,982, Charlton U.S. Pat. No. 5,714,389, Rosenstein, U.S. RE 38,430 all incorporated herein by reference.
Lateral flow assays are characterized in that a liquid solution containing an analyte to be detected is transported by capillary action laterally along a membrane strip. The membrane strip typically has reagents impregnated in the membrane. Sample is applied to one end of the strip (typically at a first absorbent pad) and sometimes with the aid of a solvent such as water. The sample may be mixed with a labeling reagent having a first binding reagent before contact with the strip, or the strip may contain labeling reagent therein. As the liquid passes through a “detection zone,” second binding reagents immobilized on the strip permit visualization of the assay results. The lateral flow assay is typically rapid and provides sensitive and accurate detection of analytes, depending in part on the selection of the binding reagents used.
Lateral flow assays may employ “competitive” or “noncompetitive” techniques, both of which are well-known in the art. In the competitive-type immunoassay, analyte in a sample is mixed with analyte that is conjugated to a detectable label. The mixture is then contacted with a lateral flow test strip. The mixture then migrates along a flow path defined by a membrane. The unlabeled analyte (from the sample) and labeled analyte compete for a limited number of binding sites on a binding agent immobilized on the test strip. The amount of labeled analyte detected at the detection region in a competitive assay is inversely proportional to the concentration of analyte in the sample (i.e., a greater amount of accumulated label indicates lower levels of analyte in the test sample).
In contrast, in “non-competitive” or “sandwich”-type immunoassays, antigen in the sample binds to a first binding reagent (such as an antibody) conjugated to a label (the “labeling reagent”). The sample containing antigen bound to the labeling reagent is then contacted with a lateral flow assay test strip. As the mixture migrates by capillary action along the membrane, the analyte-labeling reagent complex contacts and binds to a second binding reagent immobilized in the membrane. The label-analyte complex accumulates on the membrane, and a visible indicator line results. The amount of accumulated label is directly proportional to the concentration of the antigen in the sample. Both competitive-type and non-competitive-type assays are described in Ching et al, U.S. Pat. No. 6,534,320, incorporated herein by reference.
The lateral flow immunoassays typically employ the same basic components. These are described in, for example, Ching et al, U.S. Pat. No. 6,534,320 and May et al. U.S. Pat. No. 6,228,660. These components are: a first absorbent material, a membrane (such as nitrocellulose), and a second absorbent material, wherein the test strip has reagents impregnated therein for the detection of analytes.
Lateral flow devices can also be categorized as using either a one-step or two-step method. The two-step method (also referred to as the “pour on” method) is described in European Patent Application 0 250 137 A2, entitled “Colloidal Gold Immunoassay,” published Dec. 12, 1987 (“Mochnal”). In this method, the sample and labeling reagent are mixed prior to contacting the sample with the lateral flow test strip. After mixing sample with labeling reagent, the mixture is contacted with a first absorbent material to initiate the lateral flow assay. The sample then flows along the membrane, contacting one or more immobilized second binding reagents. Analyte in the sample binds to the second binding reagent and accumulated label results in a visible reaction. The two-step method is characterized by the initial step of pre-mixing liquid sample with labeling reagent prior to contacting the mixture to the test strip.
In contrast, in the “dried-on” or “one-step” method, sample is not mixed with labeling reagent prior to contacting a test strip. In the one-step method, the labeling reagent is pre-dried and embedded within the test strip, typically within the first absorbent pad. Liquid sample applied directly to the first absorbent pad solubilizes the dried labeling reagent. As the liquid sample flows laterally along the test strip towards the test site, analyte binds to and transports the labeling reagent bound to analyte to an immobilized second binder. As in the two-step method described above, the analyte reacts with a second binding reagent immobilized on the matrix to effect a visual result. The one-step method is distinct from the two-step method primarily in that all of the reagents necessary for the assay are present in dry form on the test strip, eliminating the need for a separate mixing step.
Additionally, cross-contamination and sanitation is often a concern in the use of lateral flow assays. Test strips used for detection of analytes in biological samples, such as urine, saliva or feces, pose a potential contamination hazard when the test strips are contacted with sample and then transported to a different location. Contamination can occur when the test strips are in use, or upon disposal of the strips. As such, it is desirable to have a device that provides sanitary handling and disposal, minimizing cross contamination of test strips or personnel.
The invention described herein provides a support for a test strip, in particular, a lateral flow immunoassay test strip, and a device for conducting assays using test strips, that provide for improved ease of use, assembly, sanitary handling and disposal. The invention further relates to a device that may be used for detection of multiple labeling reagents including those that emit light or that require the use of instrumentation such as spectrophotometers.